芒果苷抑制中波紫外线诱导人成纤维细胞提前衰老的相关研究

Mangiferin inhibits ultraviolet B-induced premature senescence in human fibroblasts： an experiment study

目的研究芒果苷是否对反复亚毒性剂量中波紫外线（UVB）诱导的人二倍体成纤维细胞的早衰起抑制作用及其可能的机制。方法实验分成空白对照组（不做处理），UVB-应激诱导的提前衰老（sIPS）组（单纯照光），芒果苷4．0mg／L组（单纯加药），UVB＋芒果苷1．0mg／L组、UVB＋芒果苷2．0mg／L组、UVB＋芒果苷4．0mg/L组（UVB照射联合药物处理）。成纤维细胞经5次UVB10mJ/cm2照射后，用细胞计数法（CCK8法）检测细胞增殖活性，B半乳糖苷酶染色（SA—β—Gal）计算衰老细胞百分比，流式细胞仪测定细胞周期，蛋白印迹检测衰老相关蛋白p53、p21、p16含量，实时荧光定量PCR（RT—PCR）检测基质金属蛋白酶1（MMP1）、基质金属蛋白酶3（MMP3）、I型胶原、Ⅲ型胶原mRNA和衰老相关基因p53、p21、p16mRNA表达。采用单因素方差分析进行数据统计分析。结果UVB＋芒果苷1．0mg／L组、UVB＋芒果苷2．0mg／L组、UVB＋芒果苷4．0mg／L组以及UVB—SIPS组细胞增殖活性（A450）依次为0．3229±0．0113、0．3361±0．0163、0．3426±0．0144、0．2882±0．0207（F=110．08，P〈0．05），B半乳糖苷酶染色阳性率依次为（88．83±4．54）％、（46．33±5．51）％、（32．17±6．05）％、（93．67±3．75）％（F=283．54，P〈0．05；与UVB—SIPS组比较，除UVB＋芒果苷1．0mg／L组外，其他两组均P〈0．05）；G1期细胞阻滞率依次为（72．19±3．42）％、（60．99±2．70）％、（49．80±2．10）％、（82．09±0．89）％（F=156．01，P〈0．05）。与UVB—SIPS组相比，UVB＋各浓度芒果苷组细胞MMP1和MMP3mRNA表达降低（F=69．41、106．41。均P〈0．05），I型胶原和Ⅲ型胶原mRNA表达增加（F=66．41、46．81，除UVB＋芒果苷1．0mg／L组P〉0．05外，其他各组均P〈0．05），衰老相关基因p53、p21、p16mRNA表达降低（F=265．60、151．82、329．85，均P〈0．05），衰老相关蛋白p53、p21和p16的合成减少（F=160．51、158．53、75．38，均P〈0．05），各指标检测值的变化与芒果苷浓度之间呈一定依赖性。结论芒果苷可能通过下调p53、p21、p16基因的表达来抑制UVB诱导的人二倍体成纤维细胞的早衰。

Objective To clarify whether mangiferin inhibits stress-induced premature senescence （SIPS） in human diploid fibroblasts （HDFs） induced by repeated exposure to a sub-cytotoxic dose of ultraviolet B （UVB）, and to investigate the mechanism underlying the effect of UVB. Methods HDFs were isolated from the circumcised foreskin of healthy males, and subjected to a primary culture and 6 - 12 passages of subculture. Then, the HDFs were divided into six groups, i.e. blank control group receiving no treatment, UVB-SIPS group receiving UVB irradiation only, three combination groups receiving UVB irradiation and post-irradiation treatment with mangiferin at 1.0, 2.0 and 4.0 mg/L respectively, and mangiferin group treated with mangiferin 4.0 mg/L only. Irradiation was performed once a day for five consecutive days, and mangiferin treatment was given immediately after each irradiation. Cell counting kit-8 （CCK-8） was used to evaluate the proliferative activity of cells, and β-galactosidase （SA-β-Gal） staining to estimate the degree of premature senescence in cells, flow eytometry to detect cell cycle, Western blot to quantify the protein expressions of senescence-related proteins p53, p21 and p16, real time-PCR to determine the mRNA expression levels of matrix metalloproteinase （MMP1）, MMP3, collagen types I and III, p53, p21 and p16, at 72 hours after the last irradiation. One-way analysis of variance was used for statistical analysis. Results The proliferative activity （expressed as the absorbance value at 450 nm） of HDFs was 0.322 9 ± 0.011 3,0.336 1 ± 0.016 3, 0.342 6 ± 0.014 4 and 0.288 2 ± 0.020 7 respectively in the UVB ± mangiferin 1.0 mg/L group, UVB ± mangiferin 2.0 mg/L group, UVB ± mangiferin 4.0 mg/L group, and UVB-SIPS group respectively （F = 110.08, P 〈 0.05）, with the percentage of SA-β-Gal-positive cells being （88.83 ± 4.54）%, （46.33 ± 5.51）%, （32.17 ± 6.05）% and （93.67 ± 3.75）% respectively （F = 283.54, P 〈 0.05）, and the proportion of cells in the G1 phase being （72.19 ± 3.42）%, （60.99 ± 2.70）%, （49.80 ± 2.10）% and （82.09 ± 0.89）% respectively （ P = 156.01, P 〈 0.05）. Compared with the UVB-SIPS group, the three combination groups showed significantly decreased expression levels of MMP1 and MMP3 mRNAs （F = 69.41, 106.41, respectively, both P 〈 0.05） as well as p53, p21 and p16 mRNAs （ F = 265.60, 151.82, 329.85, respectively, all P 〈 0.05） and proteins （ F = 160.51, 158.53, 75.38, respectively, all P 〈 0.05）, but increased expression levels of COLlal and COL3al mRNAs （ F = 66.41, 46.81, respectively, both P 〈 0.05）. In these combination groups, the changes in the above parameters were dependent on the concentration of mangiferin to a degree. Conclusion Mangiferin may inhibit UVB-induced premature senescence in HDFs via downregulating the expressions of p53, p21 and p16 genes.